Graft copolymers and graft copolymer/protein compositions

ABSTRACT

Graft copolymers comprising a poly-alpha-olefin base polymer selected from the group consisting of polyethylene, polypropylene, polystyrene, and compatible mixtures thereof, having grafted thereto an olefinic monomer. The grafted monomer is present in an amount effective to increase the amount of protein that will bind to the graft copolymer as compared with the base polymer. Also disclosed are polymer/protein compositions comprising a graft copolymer having a protein immobilized on the surface thereof, processes for the preparation of the above-described graft copolymers and compositions, methods of immobilizing proteins, and methods of immunoassay based on such immobilization.

This is a division of application Ser. No. 08/071,327, filed Jun. 2,1993 and now U.S. Pat. No. 5,364,907 which application is a continuationof Ser. No. 07/595,275, filed Oct. 10, 1990 and now abandoned.

BACKGROUND OF THE INVENTION

Technical Field

This invention relates to graft copolymers and processes for theirpreparation. In another aspect, this invention relates to immobilizationof proteins on synthetic polymers and also to methods of immunoassaybased on such immobilization. This invention also relates to polymerswith proteins immobilized on the surface thereof.

Description of the Related Art

Processing and/or production of polymers using wiped-surface reactorssuch as screw extruders and twin-screw extruders is well known (suchprocessing is often referred to as reactive extrusion). Twin-screwextruders and their use in continuous processes such as graftpolymerization, alloying, bulk polymerization of vinyl monomers, andcondensation and addition reactions are generally described in PlasticsCompounding, January/February 1986, pp. 44-53 (Else et al.) and PlasticsCompounding, September/October 1986, pp. 24-39 (Frund et al.). Graftreactions are said to be carried out by first melting a polymericspecies in the initial stages of an extruder, injecting a peroxidecatalyst into the extruder, and mixing in a monomer under high shearconditions. Advantages of the twin-screw extrusion process are said toinclude narrow distribution of molecular weight, improved melt-flowproperties, consistent process control, and continuous processing.

Graft polymerization reactions of polyolefins with various monomersusing wiped-surface reactors are known. Such grafting is said to beuseful in providing a polymer adduct with functionality to allow furthermodification of structure and properties, and general mechanisticproposals regarding the formation of these "mechanochemicallysynthesized" adducts are discussed in connection with the grafting ofmaleic anhydride onto polypropylene in Polymer Prep., 1986, 27, 89(Al-Malaika). Particular free radical graft polymerization reactionshave been reported. For example, U.S. Pat. No. 3,177,270 (Jones et al.)discloses a process of preparing graft copolymers by malaxing an olefinpolymer at a temperature between 110° C. and 250° C. while contactingthe polymer with a minor proportion of a mixture comprising a monovinylaromatic compound and optionally one or more other monomers such asacrylic acid, methacrylic acid, acrylonitrile, methyl methacrylate,methacrylonitrile, or maleic anhydride, the mixture having dissolvedtherein an organic peroxide. British Pat. No. 1,393,693 (Steinkamp etal.) discloses the use of a single-screw extruder to graft monomers suchas maleic anhydride and acrylic acid onto polyolefins such aspolypropylene in the presence of a suitable free radical initiator suchas an organic peroxide. The product graft copolymers are said to have amelt flow rate (MFR) of at least 50% greater than the MFR of the basepolymer.

U.S. Pat. No. 4,003,874 (Ide et al.) discloses modified polyolefinsobtained by adding an unsaturated carboxylic acid or an anhydridethereof and an organic peroxide to a polyolefin and melting thesecomponents in an extruder. The polyolefin so obtained adheres to glassfibers.

U.S. Pat. No. 4,146,529 (Yamamoto et al.) discloses a process for theproduction of modified polyolefins by combining a polyolefin with one ormore carboxylic acids or their anhydrides in the presence of a radicalproducing agent in an extruder and in the presence of an organosilane.

U.S. Pat. No. 4,228,255 (Fujimoto et al.) discloses a method forcrosslinking a polyolefin, the polyolefin being a low densitypolyethylene or a polyolefin mixture containing a low densitypolyethylene, comprising reacting the polyolefin with an organic silaneand an organic free radical initiator to form a silane-graftedpolyolefin, then mixing the silane-grafted polyolefin with a silanolcondensation catalyst. The mixture is extruded with heating in asingle-screw extruder to obtain a crosslinked polyethylene.

Among the myriad properties of some synthetic polymers is their abilityto reversibly bind proteins. Many techniques for assay ofprotein-containing substrates are based on such binding. Enzyme linkedimmunosorbent assay, described in "Biomedical Applications ofImmobilized Enzymes", Vol. 2, T. M. S. Chang, Ed. Plenum PublishingCorp., (Engvall) is but one such technique. ELISA and other enzymeimmunoassay techniques such as those described in Clin. Chem. 1976, 22,1243 (Wisdom) generally use a material such as glass, polycarbonate, orpolystyrene as a solid-phase immune adsorbent, which immobilizes onemember of an immunological pair. The subsequent assay relies oncompetitive binding of the other member of the immunological pair inlabeled and unlabeled form, to the immobilized member. One recognizeddisadvantage of the use of such techniques is that the immobilizedprotein is only physically adsorbed to the immune adsorbent such thatadsorbed protein can be washed off to various degrees by rinsing orcontact with aqueous buffer solutions. A decrease in assay accuracy,precision, and sensitivity can result from such "leakage" of theadsorbed protein.

SUMMARY OF THE INVENTION

This invention provides graft copolymers comprising a poly-alpha-olefinbase polymer selected from the group consisting of polyethylene,polypropylene, polystyrene, and a compatible mixture of any two or morethereof, having grafted thereto an olefinic monomer selected from thegroup consisting of: in the instance of a polyethylene base polymer,1-vinylimidazole, polyethylene-glycol monomethacrylate, andN-vinylpyrrolidone, and a mixture of any two or more thereof; in theinstance of a polypropylene base polymer, 1-vinylimidazole; in theinstance of a polystyrene base polymer, 1-vinylimidazole; and in theinstance of a base polymer mixture of any two or more of polyethylene,polypropylene, and polystyrene, 1-vinylimidazole;

the grafted monomer being present in an amount effective to increase theamount of protein that will bind to the graft copolymer as compared withthe base polymer.

This invention also provides a polymer/protein composition comprising: agraft copolymer that comprises a poly-alpha-olefin base polymer selectedfrom the group consisting of polyethylene, polypropylene, polystyrene,and a compatible mixture of any two or more thereof, having graftedthereto an olefinic monomer selected from the group consisting of: inthe instance of a polyethylene base polymer, 1-vinylimidazole,hydroxyethyl methacrylate, N,N-dimethylacrylamide, polyethyleneglycolmonomethacrylate, N-vinylpyrrolidone, and a mixture of any two or morethereof; in the instance of a polypropylene base polymer,1-vinylimidazole; in the instance of a polystyrene base polymer,1-vinylimidazole; and in the instance of a base polymer mixture of anytwo or more of polyethylene, polypropylene, and polystyrene,1-vinylimidazole; the grafted monomer being present in an amounteffective to increase the amount of protein that will bind to the graftcopolymer as compared with the base polymer, with a protein immobilizedon the surface of said composition.

This invention also provides processes for preparing the graftcopolymers described above. One such process comprises the steps of:

1) feeding to a reactor materials comprising

(a) the poly-alpha-olefin base polymer

(b) an effective amount of a free radical initiator system comprisingone or more free radical initiators; and

(c) the olefinic monomer; wherein all materials are substantially freeof oxygen;

2) reacting the materials in the reactor to provide a graft copolymer asdescribed above; and

3) withdrawing the graft copolymer from the reactor.

This invention also provides a method of immobilizing a protein,comprising the step of:

contacting the protein with a graft copolymer surface, wherein the graftcopolymer comprises a poly-alpha-olefin base polymer selected from thegroup consisting of polyethylene, polypropylene, polystyrene, and acompatible mixture of any two or more thereof, having grafted thereto anolefinic monomer selected from the group consisting of: in the instanceof a polyethylene base polymer, 1-vinylimidazole, hydroxyethylmethacrylate, N,N-dimethylacrylamide, polyethyleneglycolmonomethacrylate, N-vinylpyrrolidone, and a mixture of any two or morethereof; in the instance of a polypropylene base polymer,1-vinylimidazole; in the instance of a polystyrene base polymer,1-vinylimidazole; and in the instance of a base polymer mixture of anytwo or more of polyethylene, polypropylene, and polystyrene,1-vinylimidazole, the grafted monomer being present in an amounteffective to increase the amount of protein that will bind to the graftcopolymer as compared with the base polymer, at a temperature and for atime sufficient to cause the protein to become immobilized on thesurface.

Further, the invention provides a method of immunoassay comprising thesteps of:

1) treating an article comprising a surface of a polymer/proteincomposition as described above with one member of an immunological pair;

2) incubating the treated article with a solution suspected ofcontaining the second member of the immunological pair; and

3) determining the amount of the second member of the immunological pairpresent in the solution.

By virtue of the grafted monomers, graft copolymers of the inventionprovide an increased amount of irreversible binding (i.e., immobilizing)of proteins for the purposes of, e.g., immunoassay. Accordingly, graftcopolymer/protein compositions of the invention allow improvement inbioassay accuracy, precision, and sensitivity.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exemplary flow diagram of a process for preparing the graftcopolymers of the invention and those used in the compositions of theinvention. Ancillary equipment known to those skilled in the art, suchas pumps and valves, has not been illustrated, and secondary processstreams such as utility lines (e.g., cooling water) have been omitted.

FIG. 2 is a flow diagram of a counter-rotating twin screw extruderuseful in preparing graft copolymers.

DETAILED DESCRIPTION OF THE INVENTION

A graft copolymer of the invention comprises a poly-alpha-olefin basepolymer and a monomer grafted thereto via an alkenyl group. The doublebond is of course not present in the product graft copolymer; rather, inthe grafting process the alkenyl group becomes a saturated (e.g.,alkylene) link between the base polymer and the grafted moiety. In theinstant specification and claims a reference to a grafted alkenyl groupdesignates such a saturated link and does not designate the presence ofolefinic unsaturation in the grafted monomer as it is incorporated inthe graft copolymer.

Suitable base polymers include poly-alpha-olefins selected from thegroup consisting of polyethylene, polypropylene, polystyrene, and acompatible mixture of any two or more thereof. Base polymers ofvirtually any molecular weight are suitable. Likewise, base polymers andcompatible mixtures thereof with a wide range of melt index values(e.g., from about 0.1 to about 1500) are suitable.

The olefinic monomer is selected from the group consisting of: in theinstance of a polyethylene (PE) base polymer, 1-vinylimidazole (VIm), apolyethylene glycol monomethacrylate (PEG; polyethyleneglycolmonomethacrylates of virtually any molecular weight, e.g., in the rangefrom about 200 to about 10,000 are suitable), N-vinylpyrrolidone (NVP),and a mixture of any two or more thereof; in the instance of apolypropylene (PP) base polymer, 1-vinylimidazole; in the instance of apolystyrene (PS) base polymer, 1-vinylimidazole; and in the instance ofa base polymer mixture of any two or more of polyethylene,polypropylene, and polystyrene, 1-vinylimidazole.

A graft copolymer of the invention comprises an amount of the graftedmonomer effective to increase the amount of protein that will bind tothe graft copolymer as compared with the base polymer. Stated anotherway, the graft copolymer binds proteins to a greater degree than doesthe base polymer. The amount that constitutes an effective amount of thegrafted moiety will depend upon the particular grafted monomers and theparticular base polymer. Generally, however, a graft copolymer comprisesabout 0.01% to about 20%, preferably 0.5 to about 10% by weight ofgrafted monomer. In the preparation of the graft copolymers (describedin detail below) it is preferred to use like quantities of monomer,i.e., preferably about 0.01 to about 20% or more by weight, morepreferably 0.5 to about 10% by weight based on the weight of the basepolymer.

In a graft copolymer/protein composition of the invention, differentgrafted monomers are suitable depending on the particular base polymer.In the instance of a polyethylene base polymer, the grafted monomer isselected from the group consisting of 1-vinylimidazole, hydroxyethylmethacrylate (HEMA), N,N-dimethylacrylamide (DMA), polyethyleneglycolmonomethacrylate, N-vinylpyrrolidone, and a mixture of any two or morethereof. In the instance of a polypropylene or polystyrene base polymer,and in the instance of a compatible base polymer mixture of any two ormore of polyethylene, polypropylene, polystyrene, the grafted monomer is1-vinylimidazole.

A graft copolymer used in a graft copolymer/protein composition of theinvention, like a graft copolymer of the invention, comprises an amountof the grafted monomer effective to increase the amount of protein thatwill bind to the graft copolymer as compared with the base polymer. Theamount that constitutes an effective amount is as discussed above inconnection with graft copolymers of the invention.

In order to prepare a graft copolymer, the base polymer and the monomerare reacted in the presence of an initiator system comprising one ormore free radical initiators. The initiator system serves to initiatefree radical grafting of the monomer. In a process involving a basepolymer that does not undergo substantial crosslinking under polymermelt conditions in the presence of a free radical initiator, the basepolymer is degraded in the reactor. However, the selection of anappropriate initiator system affords a product graft copolymer thatbetter retains the molecular weight of the base polymer.

Many initiators are known. Suitable initiators include: hydroperoxidessuch as cumene, t-butyl, and t-amyl hydroperoxides, and2,5-dihydroperoxy-2,5-dimethylhexane; dialkyl peroxides such asdi-t-butyl, dicumyl, and t-butyl cumyl peroxides,2,5-dimethyl-2,5-di(t-butylperoxy)hexane, and2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne; peroxyesters such ast-butyl perbenzoate and di-t-butyl-diperoxy phthalate, diacyl peroxidessuch as benzoyl peroxide and lauroyl peroxide; peroxyketals such asn-butyl-4,4-bis(t-butylperoxy)valerate and1,1-di-t-butylperoxy-3,3,5-trimethylcyclohexane; and azo compounds suchas azoisobutyronitrile.

The reaction conditions under which a graft copolymer is preparedtypically involve heating at about 150° C. to about 250° C. Thereactants typically have a residence time of about 1 to about 20 min. Itis therefore difficult to select a single initiator with a decompositionrate such that initiating radicals will be present in a substantialconcentration for a prolonged period of time when a relatively lowconcentration of initiator is used. It is therefore preferred to use amixture of at least two initiators as an initiator system.

Proper selection of the components of the initiator system overcomes theabove-discussed difficulty with single initiators, and allows controland optimization of the physical properties of the product graftcopolymer. Generally it is preferred that each initiator in an initiatorsystem have a rate of decomposition substantially different from thoseof the other initiators in the initiator system. For example, in aprocess with a residence time of about 5-10 minutes at a temperature ofabout 200° C., an initiator system wherein one initiator has a half-lifeof about 30 seconds and the other initiator has a half-life of about 2minutes has been found to be suitable.

Preferred initiator systems include mixtures comprising from about 40%to about 60% by weight of 2,5-dimethyl-2,5-di(t-butylperoxy)hexane,(such as that commercially available as LUPERSOL™ 101 from PennwaltCorporation) and from about 60% to about 40% by weight of an initiatorsuch as 2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne, (such as thatcommercially available as LUPERSOL™ 130 from Pennwalt Corporation),t-butylhydroperoxide, or di-t-butylperoxide. Initiator decompositionrates are temperature dependent, and other particular initiator systemsand preferred concentration thereof can be selected by those skilled inthe art consistent with the temperature of the reaction and theresidence time of the reactants.

The total initiator concentration is preferably from about 0.1% to about1%, more preferably from about 0.25% to about 0.5% based on the weightof the base polymer.

The graft copolymers can be prepared using various well known reactorssuch as stirred tank reactors, tubular reactors, and extruders. Thegraft copolymers are preferably made by a process involving awiped-surface reactor. A wiped-surface reactor comprises a shell orvessel that contains at least one rotor having a wiping portion locatedclose to the inside surface of the shell and a root portion that isspaced substantially further from the shell than the wiping portion. Asthe rotor is rotated, the wiping portion passes close enough to theinside surface of the shell to clean the surface and form a seal whenthe reactor contains monomer and/or polymer but not so close as to causepermanent deformation of either the rotor or shell. It is necessary thatthe root surface of the rotor also be wiped or cleaned continuouslyduring the operation of the reactor.

Intermeshing twin screw extruders can be used as wiped-surface reactors.The screws function as the rotors and the flight lands function as thewiping portion, while the screw root surface between the flight landsfunctions as the root surface. Clearances between the inside of thebarrel wall of the extruder and the flight lands of the screws arepreferably in the range of about 0.25 to 0.5 mm. Although co-rotatingtwin screw extruders can be used, counter-rotating twin screw extrudersare preferred. The counter-rotating extruder acts as a positivedisplacement pump conveying the reactant stream, and it also behaveslike a series of small mixing zones or continuous stirred tank reactors.The counter-rotating twin screw extruder also gives good control overmelting, mixing, and reaction temperatures.

Preferably, the screws of a counter-rotating twin screw extruder aredivided into segments, i.e., the extruder screws can be composed of anumber of separate screw segments that fit onto a common drive shaft bymeans of a keyway and can be disassembled and rearranged in variousorders and configurations. It is also possible to utilize screw segmentshaving multiple (e.g., two or three) starts and various pitch, and oneor more screw segments can be reversed in order to increase mixing.Residence time of the reactants, and thus the properties of theresultant product, can therefore be varied by selection of screw pitchand/or screw speed (i.e., screw rpm). Furthermore, each particular zoneof a twin screw extruder can be independently heated or cooled byexternal heating or cooling means, allowing further control of reactionconditions.

The use of a wiped-surface reactor is discussed with reference toFIG. 1. The base polymer can be fed in a region of the reactorcoincident with the region in which the initiator system is fed. Forexample, the desired base polymer, preferably in pellet form, can bewetted with a free radical initiator system and purged with an inert gassuch as nitrogen, helium, argon or the like, to render the materialsubstantially free of oxygen (i.e., oxygen, if present, is present in anamount such that it does not significantly affect the desired freeradical polymerization reactions). It is preferred to carry out thereaction under anhydrous conditions.

The base polymer/initiator mixture can be fed at a predetermined rateinto feed zone 1 of the wiped-surface reactor. It is preferred, however,to feed the base polymer in a region of the reactor prior to orcoincident with the region in which the initiator system is fed.Preferably, in instances where the base polymer is a poly-alpha-olefinthat does not undergo substantial crosslinking under polymer meltconditions in the presence of a free radical initiator, the base polymeris fed to the reactor in a region of the reactor preceding or coincidentwith the region in which the initiator system is fed, and the monomer isfed to the reactor in a region of the reactor subsequent to the regionin which the initiator is fed. In instances where the poly-alpha-olefinbase polymer undergoes substantial crosslinking under polymer meltconditions in the presence of a free radical initiator, the base polymerand the initiator are preferably fed to the reactor in a regionpreceding the region in which the monomer is fed, but at a temperaturesuch that crosslinking of the base polymer is minimized or preventedprior to the addition of the monomer.

The feed zone 1 typically comprises a feed throat, into which the basepolymer can, if desired, be fed into the upstream end, and into whichthe initiator system can be fed at the downstream end. A furtheralternate method of feeding the base polymer and the initiator involvesthe use of a 2-component feed zone consisting of a base polymer feedzone into which the base polymer is fed, followed in sequence by aseparate initiator feed zone into which the initiator is fed. Theextruder is preferably starve fed, i.e., all material fed into the feedzone is conveyed into initiation/melt zone 2 of the extruder, andnothing is held up in the feed zone 1. Feed rates can vary with the sizeof the reactor and for any given size of reactor, one skilled in the artwill be able to determine suitable feed rates. As an example, when aLEISTRITZ™ 34 mm counter-rotating twin screw extruder is used feed ratesare preferably from about 0.4 Kg/h to about 9 Kg/h. The feed zone screwpreferably has a high pitch (e.g., 20 mm) to accommodate base polymerpellets. The feed zone can, if desired, be operated in a temperaturecontrolled manner, depending on the reactants, reaction conditions andthe like. Generally, it is suitable to maintain the feed zone of theextruder in a temperature range from about 10° C. to about 50° C.,depending on the base polymer used.

In initiation/melt zone 2, the initiator system and the base polymer aremixed and heated. When non-crosslinking base polymers such aspolypropylene and polystyrene are used, the temperature is preferablysuch that radical chain reactions are initiated. Preferred temperatureswill depend on the particular base polymer and initiator system, butgenerally temperatures in the range between 150° C. and about 250° C.are suitable. When crosslinking base polymers such as polyethylene areused, both the feed zone and the initiation/melt zone are preferablykept at a temperature such that the initiator does not produceinitiating radicals at a significant rate. As the residence time of thematerials in these zones is only a small fraction of the total residencetime, this serves to minimize or prevent the crosslinking of the basepolymer prior to addition of the monomer. Again preferred temperatureswill depend on the particular base polymer and initiator system.Generally, however, temperatures between about 100° C. and 150° C. arepreferred.

In monomer addition zone 3, a nitrogen-purged monomer is added, usuallyby means of a high pressure pump and under an inert atmosphere. Themonomer is generally fed as a liquid or as a solution in an inertsolvent (e.g., decane, toluene, tetrahydrofuran or the like). Again,feed rates are variable, and when a LEISTRITZ™ 34 mm counter-rotatingtwin screw extruder is used, feed rate is preferably about 4 g/h toabout 180 g/h. It is preferred to maintain the monomer addition zone ata temperature of about 150° C. to about 250° C.

Grafting proceeds in reaction zone 4. The reaction zone is heated. Thepreferred temperature will depend on the particular base polymer andinitiator system used. Further, the preferred temperature of thereaction zone will depend on the intended residence time in the reactionzone. Generally, temperatures in the range of 150° C. to 250° C. andresidence times in the range of 1 minute to 10 minutes are suitable.

In reactions where there remains residual monomer, it is preferred toremove the residual monomer from the product by venting. This can bedone in devolatilization zone 5, where a vacuum (e.g., about 10 kPaabsolute pressure) can be applied to a vent line. The resultant productis passed through block zone 6, which conveys the product graftcopolymer for any further processing as desired, e.g., shaping in a die,extruding, quenching in a suitable quenching liquid, or pelletizing touseful dimensions for convenience of handling and/or storage.

In instances where it is desirable to quench the graft copolymer in aquenching liquid, any suitable quenching liquid can be used. Water iscommonly used. However, quenching in water can cause some undesirablehydrolysis of grafted hydrolytic moieties (if any), such as esters thatwill be present in graft copolymers wherein polyethyleneglycolmonomethacrylate or hydroxyethyl methacrylate are the grafted monomers.Further, quenching in water can cause the graft copolymer to have arelatively high moisture content, which can cause internal hydrolysis ofhydrolytic groups (if any) and poor performance of the graft copolymerupon molding. Therefore, it is preferred to quench the graft copolymerin a quenching liquid that is inert to any functional groups present inthe monomer. It is also desirable for such a quenching liquid to havelow volatility and a high specific heat. Suitable quenching liquids canbe easily selected by those skilled in the art. Particularly preferredquenching liquids include inert liquid fluorocarbons.

A graft copolymer surface can bind (i.e., immobilize) proteins. Theprotein can be, for example, an antibody such as anti-human IgE, aprotein such as Protein A, or an enzyme. Preferred proteins forimmobilization include those with a molecular weight of at least 1000,most preferably at least about 4000.

A graft copolymer can be prepared, for example, in the form of anarticle such as a microtiter well or a test tube or in the form of beadsor a film. To bind (i.e., immobilize) a protein to the surface of thearticle, the article can be contacted, e.g., incubated, with a protein,e.g., a serum or other solution containing a protein. The protein canalso, if desired, contain a trace level of labeled (e.g., radiolabeledor fluorescence-labeled) protein to allow assay of the protein. Anarticle with a protein bound thereto can then be further incubated, forexample, with a relatively concentrated second protein solution such asbovine serum albumin, to block any remaining surface of the article andto displace initially adsorbed protein from the surface of the article.

An article treated as described above can be treated (e.g., incubated)with a protein denaturing agent such as sodium dodecylsulfate (SDS) toremove loosely-bound protein from the surface. Analysis of the resultingarticle shows that the amount of protein that is retained on the graftcopolymer surface is increased by the grafted moiety.

The increased amount of irreversible binding of proteins such asantibodies in the graft copolymer/protein compositions of the inventionsuggests utility in applications where protein immobilization isdesirable, e.g., diagnostic applications in which proteins areimmobilized, including microtiter well assay devices, bead suspensions,and the like for use in ELISA and other well known enzyme immunoassaytechniques such as those described in Clin. Chem. 1976, 22, 1263(Wisdom). Furthermore, it is known that a proteinaceous layer willpromote binding of cells to hydrophobic and hydrophilic base polymers.This invention allows one to immobilize proteins such as albumins,collagens, basement membrane fractions, etc., or specific proteins suchas fibronectin, laminin, monoclonal antibodies, or adhesion proteins,etc., all of which can promote binding of cells to a polymer surface.

The immobilization of a protein on a graft copolymer can be carried outby contacting the protein with a graft copolymer surface at atemperature and for a time sufficient to cause the protein to bind tothe graft copolymer surface. While it is not practical to enumerateparticular conditions suitable for each and every protein, suchconditions can be easily selected by those skilled in the art.Generally, however, room temperature exposure of a graft copolymersurface to a solution of the protein in an appropriate solvent will besuitable to bind the protein to the surface.

The amount of grafted moieties on the surface of a graft copolymer canbe measured by conventional means such as x-ray photoelectronspectroscopy, Fourier transform infrared spectrophotometry, attenuatedtotal reflectance infrared spectrophotometry, and the like.

The following describes the preparation of graft copolymers and graftcopolymer/protein compositions. Temperatures are in degrees Celsius, andall parts and percentages are by weight. Graft copolymers are designatedherein by enumerating the base polymer and the grafted monomer, e.g.,the designation PE/DMA represents a graft copolymer comprising apolyethylene (PE) base polymer having N,N-dimethylacrylamide (DMA)grafted thereto.

Intermediate A

Preparation of polyethylene (PE)/hydroxyethyl methacrylate (HEMA).

HEMA was grafted onto linear low-density PE (DOWLEX™ 2517, meltindex:25, Dow Chemical Co., Midland, Mich.) using a counter-rotating 34mm LEISTRITZ™ twin-screw extruder model LSM 30.3466, (Nuremburg,Germany), with a length:diameter ratio at 35:1, configured as describedbelow with reference to FIG. 2.

FIG. 2 shows a twin-screw extruder with a feed hopper 10, feed zone 12,and a heated barrel that comprises: an initiation/melt zone comprisingbarrel section 14; a reaction zone comprising a monomer feed zone(barrel section) and barrel sections 18, 20, 22, 24, and 26; adevolatilization zone comprising barrel section 28; and a block zonecomprising barrel sections 30 and 32. Each barrel section is 120 mmlong, and the extruder has a total length of 1200 mm.

Transducer ports (e.g., T4 represents transducer number 4 located inbarrel section 24) are located at 30 mm, and/or 90 mm into each heatedbarrel section. Thermocouple ports are located at 60 mm into each heatedbarrel section.

The polyethylene base polymer was directly fed into the feed throat. A1:1 mixture by weight of LUPERSOL™ 101 and LUPERSOL™ 130 was fed at 4.1mL/h, and a 1:1 mixture by weight of LUPEROX™ 500 dicumyl peroxide(Pennwalt) and decane was also fed at 4.2 mL/h, each to the feed throatand each by a separate nitrogen purged RUSKA™ pump. The HEMA was purgedwith nitrogen, added to a nitrogen-purged RUSKA™ positive displacementpump, and added at a rate of 160 mL/h in heated barrel section 16, 270mm from the start of the screws. Total flow rate was 40 g/min. Screwspeed was 103 rpm. The temperature profile was as follows: Section 14,253°; Section 16, 145°; Section 18, 181°; Section 20, 199°; Section 22,201°; Section 24, 197°; Section 26, 205°; Section 30, 234°; Section 32,205°; Section 34, 202° . In heated barrel section 28 residual monomerwas removed by vacuum. The product graft copolymer was conveyed from theblock zone (barrel sections 30 and 32), into a water bath and fed into aConair Co. (Bay City, Mich.) pelletizer to afford generally cylindricalbeads of 3-4 mm in length and about 1 mm in diameter. The melt index ofthe product graft copolymer was 18 as measured by ASTM D-1238,indicating that crosslinking of the polyethylene occurred during thegrafting process.

Intermediate B and Examples 1-3

Preparation of PE/NVP (Intermediate B), PE/VIm, PP/VIm, and PE/PEG.

PE (DOWLEX™ 2517) and PP (DYPRO™ 8771) were independently used as basepolymers, and NVP, polyethyleneglycol monomethacrylate (SIPOMER™ HEM-10Alcolac), and VIm (Aldrich Chem. Co.) were independently used asmonomers. Graft copolymers were prepared in a LEISTRITZ™ 34 mmtwin-screw extruder as generally described above in connection withIntermediate A. The monomer was purged with nitrogen gas for 15-30 minprior to use. The feed hopper and feed throat of the extruder were keptunder nitrogen gas throughout. The monomer was injected into the secondzone of the extruder via a RUSKA™ single-piston positive-displacementpump at a pressure of 50 psi. The initiator (a 1:1 mixture of LUPERSOL™101 and LUPERSOL™ 130) was fed into the open feed throat via adual-piston RUSKA™ pump. The extruded graft polymer was formed into astrand, quenched, and pelletized. Other conditions are listed in Table 1below.

                  TABLE 1                                                         ______________________________________                                        Polymerization Conditions                                                                Graft Copolymer                                                               PE/VIm PP/VIm   PE/NVP   PE/PEG                                               Ex. 1  Ex. 2    Int. B   Ex. 3                                     ______________________________________                                        Condition                                                                     Screw speed (rpm)                                                                          85       100      100    100                                     Temperature (°C.)                                                      Section                                                                       14           154      195      153    150                                     16           150      199      147    150                                     18           165      204      157    160                                     20           163      200      159    160                                     22           160      197      157    160                                     24           154      195      153    160                                     26           158      202      161    160                                     28           185      215      184    160                                     30           161      204      163    160                                     32           161      182      163    160                                     Polymer flow (g/min)                                                                       39       42.1     42.9   42                                      Init. flow (mL/h)                                                                          3.0      8.9      3.0    8.9                                     Initiator Percent                                                                          0.1      0.3      0.1    0.3                                     Monomer flow 30       120      120    160                                     (mL/h)                                                                        Percent Incorporated                                                                       0.17     3.2      1.9    3.1                                     Monomer                                                                       ______________________________________                                    

Intermediate C

Preparation of Pe/DMA.

A graft copolymer was prepared in a Brabender Plasti-corder reactor,type EPL-V5501, (C. W. Brabender Co.), equipped with a type RE.E.6mixing head, a type SP-T1002 temperature control console, and a torquerheometer. The reactor was preheated to 180° C. under a nitrogen purge.PE (DOWLEX™ 2517) was used as base polymer. N,N-Dimethylacrylamide (DMA,Aldrich Chemical Co.) was used as the monomer. The base polymer (45 g)was added to the reactor and mixed at 30 rpm until fully melted. Theinitiator (0.14 g of a 1:1 mixture of LUPERSOL™ 101/LUPERSOL™ 130) wasadded to the polymer and allowed to mix for about one-half minute. DMA(2.4 g; 5 weight percent) was added to the mixture and allowed to reactfor 3 minutes. The mixture was removed from the reactor and cooled toambient temperature. The PE/DMA contained 0.25% DMA by weight. Cooledsamples were stored in plastic bags until use.

Example 4

Preparation of PS/VIm.

According to the general method of Intermediate C, polystyrene (39 g,Polysar TM 101-300, melt index 2.2, Polysar Inc., Leominster, Mass.) and1-vinylimidazole (1.0 g) were reacted to afford PS/VIm.

Examples 5-11

The immobilization of protein on PE, PP, PS, and their graft copolymers.

Film samples of the graft copolymer with thickness of about 0.13 mm weremade by pressing (at a pressure of about 41.4 kPa for 30 seconds using aWABASH™ heated press, Wabash, Ind.) about 10 g of the graft copolymerbetween TEFLON™ plates at about 200° C. Pressed samples were quenchedfrom the molten state to the solid state in a room temperature waterbath and cut into discs of 8 mm diameter using a conventional paperpunch. Recombinant Protein A (rProtA), purchased from Repligen(Cambridge, Mass.), was radioiodinated using Iodo-beads™ (PierceChemical Co., Rockford, Ill.). rProtA (200 μL of 250 μg/mL), with aspecific radioactivity of 2000 cpm/μg of protein, was incubated withtriplicate samples of 8 mm film discs in 25 mM sodium phosphate, pH 7.5,with 150 mM sodium chloride. Incubations were terminated after 2 h byremoval of the solution followed by addition of 500 μL of 1.0Methanolamine, pH 9.0, for 1 h. Finally, the film discs were rinsed threetimes with the chloride-phosphate buffer for 45 minutes, thentransferred to a clean tube for radioactivity determination using aPackard Model 5230 Gamma Scintillation Spectrometer (Packard InstrumentCo., Downers Grove, Ill.). The film discs were subsequently incubatedfor 4 h at 37° with an aqueous solution of 1% w/v sodium dodecylsulfate(SDS), rinsed three times with the same SDS solution, followed by afinal radioactivity determination.

The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Comparative Binding of Protein on                                             Control and Grafted Polymers                                                                   Adsorbed          Tightly Bound                                               Protein   SDS     Protein                                    Example                                                                              Polymer   (μg/cm.sub.2)                                                                        Resistance                                                                            (μg/cm.sub.2)                           ______________________________________                                               PE        0.51       22%    0.11                                       5      PE/VIm    1.12      40      0.46                                       6      PE/HEMA   0.77      32      0.25                                       7      PE/DMA    0.88      27      0.23                                       8      PE/NVP    0.81      31      0.24                                              PE*       0.44      19      0.08                                       9      PE/PEG*   0.54      53      0.29                                              PP        0.90      24      0.22                                       10     PP/VIm    1.04      28      0.29                                              PS*       1.40      28      0.39                                       11     PS/VIm*   2.34      50      1.16                                       ______________________________________                                         *Protein A solution had specific radioactivity of 2250 cpm/μg         

The amount of protein bound to the base polymers increased in the orderof the increasing hydrophobicity of the base polymer, i.e., PS>PP>PE.All grafted monomers enhanced the binding of protein relative to basepolymer surfaces. All the graft copolymers had increased SDS resistancecompared with the base polymer.

Examples 12-16

Stability of protein adsorbance in the presence of blood proteins.

Protein incubation was carried out as described in Examples 5-9 above.The specific radioactivity of rProtA was 1360 cpm/μg. After the initialradioactivity determination the films were incubated for 7 days atambient temperature with 500 μL of a 1:1 buffer:human serum solution.Residual radioactivity was determined following the incubation,aspiration of the serum solution, and two buffer rinses. The films weresubjected to the SDS treatment described in Examples 5-9, and a finalradioactivity determination was made. The results are set forth in TABLE3 below.

                                      TABLE 3                                     __________________________________________________________________________    The Effect of Long-Term Incubation of                                         Protein-Bound Films in Serum                                                             Adsorbed                                                                             Post-plasma                                                                           SDS   Covalent                                      Example                                                                            Polymer                                                                             Protein                                                                              Adsorbance                                                                            Resistance                                                                          Protein                                       __________________________________________________________________________         PE    0.64 μg/cm.sup.2                                                                  0.22 μg/cm.sup.2                                                                     6%  0.04 μg/cm.sup.2                           12   PE/VIm                                                                              1.60   0.89    33    0.53                                          13   PE/HEMA                                                                             1.06   0.48    19    0.20                                          14   PE/DMA                                                                              1.04   0.41     6    0.07                                          15   PE/NVP                                                                              1.08   0.34     6    0.06                                               PP    1.18   0.39    10    0.12                                          16   PP/VIm                                                                              1.45   0.66    15    0.23                                          __________________________________________________________________________

Treatment with plasma proteins followed by SDS treatment is a stringenttest to remove proteins from a surface. Surprisingly, three of thepolymers (PE/VIm, PE/HEMA, PP/VIm) contained residual, tightly-boundprotein.

The claimed invention is:
 1. A process for preparing a uniform,homogeneous thermoplastic graft copolymer comprising the steps of:1)feeding to a reactor materials consisting essentially ofa) polyethylene,b) an effective amount of a free radical initiator system comprising twofree radical initiators, each having a decomposition rate significantlydifferent from the decomposition rates of the other initiator(s) in theinitiator system, and c) a grafted monomer selected from the groupconsisting of 1-vinylimidazole, hydroxyethyl methacrylate, polyethyleneglycol monomethacrylate, N-vinyl pyrrolidone, N,N-dimethylacrylamide andmixtures thereof effective to increase protein binding to the graftcopolymer as compared with polyethylene.
 2. A process according to claim1, wherein the initiator system comprises from about 40% to about 60% of2,5-dimethyl-2,5-di(t-butyl-peroxy)hexane and from about 60% to about40% of 2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne).
 3. A process forpreparing a uniform, homogeneous thermoplastic graft copolymercomprising the steps of:1) feeding to a reactor materials consistingessentially ofa) a base polymer selected from the group consisting ofpolypropylene, polystyrene, and mixtures thereof, b) an effective amountof a free radical initiator system comprising two free radicalinitiators, each having a decomposition rate significantly differentfrom the decomposition rates of the other initiator(s) in the initiatorsystem, and c) 1-vinylimidazole effective to increase protein binding tothe graft copolymer as compared with protein binding to only the basepolymer, wherein the base polymer is selected from the group consistingof polypropylene, polystyrene, and mixtures thereof.
 4. A processaccording to claim 3, wherein the initiator system comprises from about40% to about 60% of 2,5-dimethyl-2,5-di(t-butyl-peroxy)hexane and fromabout 60% to about 40% of 2,5-dimethyl-2,5-di(t-butylperoxy)hex-3-yne).